Novel thiopeptolide substrates for vertebrate collagenase

ABSTRACT

The disclosure relates to novel synthetic thiopeptolide substrates having high activity for the enzyme collagenase. These substrates have the following amino acid sequences: 
     
         R-Pro-X-Gly-S-Y-Z-Gly-R.sub.1 
    
     wherein 
     R=H or N-protecting group, 
     X=Leu, Ile, Phe, Val, Gln, Ala, 
     Y=Leu, Ile, Phe, Val, Ala, 
     Z=Leu, Ile, Phe, Val, Gln, Ala, and 
     R 1  =terminal amide, carboxyl or ester group.

This is a division of application Ser. No. 571,227, filed Jan. 16, 1984.

BACKGROUND OF THE INVENTION

This invention relates to novel thiopeptolides which have high activityas substrates for vertebrate collagenase.

Collagenase is a proteolytic enzyme which acts on the protein collagen.This enzyme was early found in certain clostridia culture filtrates andshown to act specifically on native (undenatured) collagen at nearphysiological pH. See Mandl, "Collagenase and Elastases," Advances inEnzymology 23, p. 163, Interscience Publishers, N.Y., 1961. Anillustrative example of a collagenase enzyme product obtained fromspecial strains of Clostridium histolyticum is commercially availablefrom Worthington Biochemical Corporation, Freehold, N.J.

Collagenolytic enzymes also have been obtained by tissue and cellculture from a wide range of animal species in which collagen ismetabolized under both physiological and pathological conditions.Collagenase enzymes from such cell and tissue culture sources or fromtissue extracts are usually obtained in exceedingly small amounts.Consequently, such enzymes are usually available only by laboratorypreparation. An illustrative example of such a preparation is a purifiedcollagenase obtained from culture media of tadpole explant as describedby Nagai et al., Biochim. Biophys. Acta 263, 564-573 (1972).

The natural substrate collagen constitutes the connective tissue of thebody and is the major type of fibrous protein in higher vertebrae,including mammals. In man, approximately one-third of the total proteincontent is collagen. The ability of collagenase to digest nativecollagen provides the enzyme with a variety of uses in tissue cultureand cell studies including the isolation of tissue collagen and othertypes of tissue dissociation. Illustratively, achilles-tendon collagenis hydrolyzed by collagenase to peptides with an average chain length offour to five amino acids.

Collagenase also is believed to be associated with the tissue invasionprocess in tumor angiogenesis, in arthritic conditions such asrheumatoid arthritis, in corneal ulceration and other diseases ofconnective tissue. It has been suggested that tumor angiogenesis factor(TAF) induces collagenase secretion by blood vessel endothelial cells.See Moscatelli et al., Cell 20, 343 (1980). The ability of TAF tostimulate collagenase production in endothelial cells provides a basisfor assay for TAF and anti-TAF. Accordingly, the measurement ofcollagenase production is a useful diagnostic tool for tissue invasion.

Conventional assays for collagenase generally are based on methodologydeveloped by Mandl et al., J. Clin. Invest. 32, 1323 (1953). Accordingto these assay procedures, collagenase is incubated for an extendedperiod of time at 37° C. with native PG,4 collagen. The extent ofcollagen breakdown is then determined using the Moore and Steincolorimetric ninhydrin method, J. Biol. Chem. 176, 367 (1948). Aminoacids which are liberated are expressed as micromoles per milligram ofcollagenase. One unit of enzyme activity equals the amount ofcollagenase required to solubilize one micromole of leucine equivalents.

Various synthetic substrates also have been developed heretofore asreagents for the quantitative determination of proteolytic enzymes suchas thrombin, plasmin, trypsin and collagenase. These substratesgenerally consist of relatively short chain peptides. Under the actionof the appropriate enzyme, a fragment is hydrolytically split off fromthe substrate with the resulting formation of a split product, thequantity of which can be measured by conventional photometric,spectrophotometric, fluorescence-photometric, and chromatographicmethods. The quantity of the split product formed per time unit is ameasure for the enzyme activity from which the quantity of enzymepresent in a given test sample can be calculated.

The following are examples of two such synthetic collagenase substrateswhich are commercially available from Peninsula Laboratories, SanCarlos, Calif., and Cal-Med, South San Francisco, Calif.:

    DNP-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH.sub.2 and

    DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH,

wherein DNP=Dinitrophenyl.

In copending applications Ser. No. 336,520, filed Apr. 8, 1982, Ser. No.450,318, filed Dec. 16, 1982 and Ser. No. 485,762, filed Apr. 18, 1983,assigned to a common assignee with this application, certain peptidesand peptolides are disclosed which have substantially greater activityas collagenase substrates than the aforesaid commercially availablesubstrates.

Other examples of peptide substrates for mammalian collagenase andmethods of measuring collagenase activity with the substrates aredescribed in U.S. Pat. Nos. 4,138,394 and 4,176,009; Nagai et al.,Biochim. Biophys. Acta 445, 521-524 (1976); Masui et al., Biochem. Med.17, 215-221 (1977); and Gray et al., Biochem. Biophys. Res. Comm.101(4), 1251-1258 (1981). Further background information on mammaliancollagenase also can be had by reference to the treatise "Collagenase inNormal and Pathological Connective Tissues," Woolley and Evanson, Eds.,John Wiley & Son, New York, 1980.

DESCRIPTION OF THE INVENTION

In accordance with the present invention novel thiopeptolides have beensynthesized as substrates for the enzyme collagenase. They havesubstantially greater activity than the aforesaid commercially availablesynthetic substrates for vertebrate collagenase and are useful in thespectrophotometric assay of the enzyme. The novel thiopeptides of thisinvention are selected from the group consisting of:

    R-Pro-X-Gly-S-Y-Z-Gly-R.sub.1                              (I)

wherein

R=H or N-protecting group,

X=Leu, Ile, Phe, Val, Gln, Ala,

Y=Leu, Ile, Phe, Val, Ala,

Z=Leu, Ile, Phe, Val, Gln, Ala,

R₁ =terminal amide, carboxyl or ester group,

and the pharmaceutically acceptable salts thereof.

The abbreviations used for the amino acids herein follow standardnomenclature in which:

Ala=L-alanine,

Gln-L-glutamine,

Gly=glycine,

Ile=L-isoleucine,

Leu=L-leucine,

Phe=L-phenylalanine,

Pro=L-proline, and

Val=L-valine.

The N-protecting groups depicted as R in the structural formula I,above, are preferably alkanoyl, aroyl, or cycloalkanoyl and morepreferably acetyl, benzoyl, carbobenzyloxy or t-butyloxycarbonyl.

The terminal groups depicted as R₁ in the structural formula I arepreferably O-alkyl, O-aryl, NH-alkyl, NH-aryl, OH and NH₂ and morepreferably OC₂ H₅ or NH₂.

The term "aryl" as used herein refers to phenyl or phenyl substitutedwith one, two or three alkyl, alkoxy, halogen, amino, hydroxy oralkanoyloxy groups.

The terms "alkyl," alkanoyl, and "alkoxy," as used herein refer togroups having 1 to 8 carbon atoms.

The term "halogen" as used herein refers to fluorine, chlorine, bromineor iodine.

The pharmaceutically acceptable salts of the thiopeptolides of thisinvention include salts of cations, such as, for example, sodium,potassium, ammonium, calcium and magnesium as well as salts derived fromeither organic or inorganic acids such as, for example, acetic, lactic,tartaric, succinic, glutaric, benzoic, salicylic, methanesulfonic,toluenesulfonic, hydrochloric, sulfuric or phosphoric acids and thelike. Desired salts can be prepared from other salts via conventionaltreatment with ion exchange resins. For non-pharmaceutical use such asin a spectrophotometric assay, the salt form need not bepharmaceutically or physiologically acceptable.

The initial peptide portion of the thiopeptolides of this invention canbe made by appropriate adaptation of conventional methods for peptidesynthesis. Thus, the peptide chain can be prepared by a series ofcoupling reactions in which the constituent amino acids are added to thegrowing peptide chain in the desired sequence. The use of variousN-protecting groups, e.g., the carbobenzoxy group (CBZ) or thet-butyloxycarbonyl group (BOC), various coupling reagents, e.g.,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),dicyclohexylcarbodiimide (DCC), carbonyldiimidazole, or1-hydroxy-benzotriazole monohydrate (HBT), and various cleavagereagents, e.g., trifluoroacetic acid, HCl in dioxane, borontris(trifluoroacetate) and cyanogen bromide, and reaction in solutionwith isolation and purification of intermediates is well-known classicalpeptide metholology.

Preferably, the peptide is prepared by the well-known Merrifield solidsupport method. See Merrifield, J. Amer. Chem. Soc. 85, 2149-54 (1963)and Science 150, 178-85 (1965). This procedure, though using many of thesame chemical reactions and blocking groups of classical peptidesynthesis, provides a growing peptide chain anchored by its carboxylterminus to a solid support, usually cross-linked polystyrene orstyrene-divinylbenzene copolymer. This method conveniently simplifiesthe number of procedural manipulations since removal of the excessreagents at each step is effected simply by washing of the polymer.

The general reaction sequence for the Merrifield peptide synthesis canbe illustrated as follows: ##STR1## This step follows cleavage of t-BOCby HCl and liberation of N-terminal amine by excess of triethylamine,thereby enabling it to react with the activated carboxyl of the nextprotected amino acid (R²). A final step involves cleavage of thecompleted peptide from the PS resin such as by anhydrous HBr in aceticacid or trifluoroacetic acid.

Further background information on the established solid phase synthesisprocedure can be had by reference to the treatise by Stewart and Young,"Solid Phase Peptide Synthesis," W. H. Freeman & Co., San Francisco,1969, and the review chapter by Merrifield in Advances in Enzymology 32,pp. 221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969. Asuitable general method of the solid phase synthesis also is describedin detail by Rivier et al, Biopolymers 17, 1927-1938 (1978).

The thiol-containing terminal portion of the novel thiopeptolides ofthis invention can be prepared by first converting an appropriate aminoacid as defined by Y in the structural formula I, above, to itsα-mercapto derivative and then coupling it to an appropriate amino acidZ-glycine ester as defined in structural formula I. The coupled productcan then be further coupled to the initial peptide portion to form thefull thiopeptolide.

For example, where amino acids Y and Z are both leucine in thestructural formula I, the thiol-containing terminal portion of thethiopeptolide can be prepared by the following general synthetic scheme:##STR2##

See, e.g., Yankeelov et al., J. Org. Chem. 43, 1623-1624 (1978) forpreparation of the L-α-mercaptoisocaproic acid by the foregoing reactionscheme. ##STR3## wherein DMF=Dimethylformamide,

HBT=1-Hydroxybenzotriazole,

EDC=1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and

TEA=Triethylamine.

Following preparation of the above thiol-containing terminal portion ofthe thiopeptolide, it can be further coupled to the initial peptideportion to form the full thiopeptolide. For example, where amino acid Xis leucine in the structural formula I, the full thiopeptolide can beprepared by the following general reaction: ##STR4##

The leucine amino acids in the foregoing general reaction schemes can bereplaced with equivalent amounts of other amino acids specified for X, Yand Z in the structural formula I, above, to provide analogousthiopeptolides in accordance with the present invention withsubstantially similar results. So also, N-protecting groups other thanacetyl and terminal groups other than ethyl ester can be employed in theforegoing general reaction schemes with substantially similar results.

The preferred thiopeptolide Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC₂ H₅ is anexcellent inhibitor (preinhibitor) of vertebrate collagenase by virtueof the fact that the enzyme cleaves this molecule to releaseHS-Leu-Leu-Gly-OC₂ H₅. The compound HS-Leu-Leu-Gly-OC₂ H₅ was found tohave an I₅₀ (median inhibitory concentration) of 10⁻⁶ M for vertebratecollagenase. The thiopeptolide is an inert molecule until it is broughtinto contact with vertebrate collagenase which effects the activation ofthe inhibitor and thereby deactivates the enzyme. Thus, thethiopeptolide when injected intravenously would be cleaved bycirculating collagenase enzyme and the fragment would then act as aninhibitor of further activity of the enzyme.

Other examples of preferred thiopeptolides have the followingstructures:

    Ac-Pro-Ala-Gly-S-Leu-Ala-Gly-OC.sub.2 H.sub.5,

    Ac-Pro-Ala-Gly-S-Leu-Phe-Gly-OC.sub.2 H.sub.5, and

    Ac-Pro-Leu-Gly-S-Leu-Phe-Gly-OC.sub.2 H.sub.5.

The high activity of the thiopeptolide as a substrate for vertebratecollagenase provides for its use in a rapid, sensitive, continuousspectrophotometric assay for the enzyme. Cleavage of the thiopeptolideby the enzyme at the thiol bond enables the determination continuouslyin the presence of 4,4'-dithiodipyridine or Ellman's reagent [DTNB,5,5'-dithiobis(2-nitrobenzoic acid)].

The reaction of the released thiol from the thiopeptolide with4,4'-dithiodipyridine or Ellman's reagent frees a chromophoric moleculewhich can be measured spectrophotometrically. For background informationon Ellman's reagent for use in colormetric assay of enzymes, see Ellman,Arch. Biochem. Biophys. 82, 70-77 (1959), and Green and Shaw, Anal.Biochem. 93, 223-226 (1979); and for such use of 4,4'-dithiodipyridine,see McRae et al., Biochemistry 20, 7196-7206 (1981).

The following specific examples will further illustrate the inventionalthough it should be understood that the invention is not limited tothese specific examples.

EXAMPLE 1 Preparation of Ac-Pro-Leu-Gly-OH

This peptide is synthesized from the component amino acids by the solidphase method as follows:

A mixture of (5.5 g, 0.031 mole) N-t-BOC-glycine, (3.6 g, 0.062 mole)potassium fluoride and (20 g, 0.0208 mole) Merrifield resin (1%crosslinked polystyrene; 200-400 mesh, 1 m.eq. chloride/gram) issuspended in 100 ml dimethylformamide (DMF) and stirred at 50° for 24hours. The resin is collected on a coarse fritted disk, washed twicewith (DMF), 50% DMF in water, 50% ethanol in water and ethanol. Thewashed resin is then dried to a constant weight in vacuo yielding 25.4 gof N-t-BOC-glycine on resin which gives a negative ninhydrin test and anamino acid analysis showing 0.714 m.mole/g attachment. See Horicki etal., Chem. Letters 165-168 (1978) for background information on thegeneral KF method of amino acid attachment to resin.

Other amino acids are added to the growing peptide chain by a series ofcoupling reactions using the desired N-t-BOC protected amino acids. Theamino acids are selected such as to prepare the following peptidesequence:

    Ac-Pro-Leu-Gly-OH

wherein Ac=acetyl.

In each case, dicyclohexylcarbodiimide (DCC) is used as the couplingagent in methylene chloride solvent. After initial coupling, the α-aminoprotecting group is removed by trifluoroacetic acid (TFA) in methylenechloride solvent followed by triethylamine (TEA) in methylene chloride.After removal of the α-amino protecting group, the remaining protectedamino acids are coupled stepwise in the aforesaid order to obtain thedesired peptide sequence. Each protected amino acid is reacted in excessDCC in methylene chloride solvent. After the amino acid sequence iscompleted, the peptide is removed from the resin support by treatmentwith anhydrous HF. At each step, excess reagents are removed by washingthe resin with methanol and/or methylene chloride solvents.

The sequence of reaction and washing steps carried out for each aminoacid addition to the growing peptide chain for preparation of theaforesaid peptides by the solid state peptide synthesis is set forth inthe following Table I.

                  TABLE I    ______________________________________    Protocol Used for Solid-State Peptide Synthesis                            Shake    Wash or Reactant        Duration    ______________________________________     1. Methylene chloride  1 min.     2. 50% TFA/methylene chloride                            1 min.     3. 50% TFA/methylene chloride                            20 min.     4. Methylene chloride  1 min.     5. Methylene chloride  1 min.     6. Methylene chloride  1 min.     7. 10% TEA/methylene chloride                            1 min.     8. Methanol            1 min.     9. 10% TEA/methylene chloride                            1 min.    10. Methylene chloride  1 min.    11. Methanol            1 min.    12. Methylene chloride  1 min.    13. Amino acid/methylene chloride                            1 min.    14. DCC/methylene chloride                            30-90 min.    15. Methylene chloride  1 min.    16. Methanol            1 min.    17. Methylene chloride  1 min.    18. Methanol            1 min.                            66-126 min.                            (1-2 hrs.)    ______________________________________

At step 14 the progress of the coupling is monitored by a ninhydrincolor test.

The crude peptide, after removal from resin by hydrogen fluoride, isextracted into water, neutralized to pH 6-7 and the water is thenremoved in vacuo. A portion of the residue is redissolved in a minimumof water and pipetted onto a 2 cm×15 cm C₁₈ reverse-phasechromatographic column. The column is washed with three column volumesof water using a Gilson peristaltic pump. A step gradient ofmethanol/water, acetonitrile/water or acetonitrile/water-pH 2.5(trifluoroacetic acid) is passed through the column to selectively elutecomponents. The eluted fractions are monitored by HPLC and fractionsrich in the desired component are pooled and lyophilized.

EXAMPLE 2 Preparation of Ac-Pro-Ala-Gly-OH

This peptide was prepared substantially similarly as Ac-Pro-Leu-Gly-OH,above, except that an equivalent amount of the N-t-BOC protected alanineinstead of leucine was used in the amino acid coupling sequence.

EXAMPLE 3 Preparation of D-α-Bromoisocaproic Acid

To a solution of D-leucine (29 g, 0.22 mol) and KBr (90 g, 0.76 mol) in450 ml 2.5N H₂ SO₄ cooled to 0° was slowly added, portionwise, NaNO₂ (23g, 0.333 mol) over a period of one and one half hours. The reactionmixture was stirred at 0° for one hour after addition, then stirred fivehours at room temperature. The organic phase was extracted into ethylether and the ether extract washed with water and dried over magnesiumsulfate. The magnesium sulfate was removed by filtration and thefiltrate concentrated in vacuo, yielding 37.5 g (87%) of crude bromide.The D-α-bromoisocaproic acid, a liquid, was twice distilled at reducedpressure through an 80 mm Vigieux column, yielding 18 g of product, bp70°-73° 0.25 mm with a consistent nmr and mass spectrum [a]_(D) ²⁰ =+42°(2, methanol)

EXAMPLE 4 Preparation of L-α-Mercaptoisocaproic Acid

To a solution of D-α-bromoisocaproic acid (17 g, 0.087 mol) in 40 mlwater adjusted to pH 5-6 with NaOH and cooled in ice was added 70 ml of2.2M sodium trithiocarbonate. The resulting solution was allowed tostand at room temperature overnight. The aqueous solution was washedwith ether, then acidified to pH 3 with H₂ SO₄. The resulting organicphase was extracted into ether and dried over magnesium sulfate. Themagnesium sulfate was removed by filtration and the filtrateconcentrated in vacuo, yielding 13.2 g of crude mercapto compound, whichwas purified by fractional distillation through an 80 mm Vigieux column,bp 86°-87° (0.9 mm). The L-α-mercaptoisocaproic acid [α]_(D) ²⁰ -31°,had a consistent nmr and mass spectrum.

EXAMPLE 5 Synthesis of L-Leucylglycine Ethyl Ester Hydrochloride

To a solution of leucylglycine (5 g, 27 mmol) in 30 ml ethanol cooled to0° was added SOCl₂ (4.1 g, 35 mmol). The reaction solution was stirredovernight at room temperature. The ethanol was removed in vacuo,yielding a glassy product with a consistent mass spectrum, nmr and asingle peak in the HPLC.

EXAMPLE 6 Synthesis of L-Penylalanylglycine Ethyl Ester Hydrochloride

This peptidyl ester hyrochloride was prepared substantially similarly asL-Leucylglycine ethyl ester hydrochloride, above, except that anequivalent amount of the dipeptide phenylalanylglycine was used insteadof leucylglycine in the esterification reaction.

EXAMPLE 7 Synthesis of L-Alanylgylcine Ethyl Ester Hydrochloride

This peptidyl ester hydrochloride was prepared substantially similarlyas L-Leucylglycine ethyl ester hydrochloride, above, except that anequivalent amount of the dipeptide alanylglycine was used instead ofleucylglycine in the esterification reaction.

EXAMPLE 8 Preparation of HS-Leu-Leu-Gly-OC₂ H₅

To a solution of L-α-mercaptoisocaproic acid (1 g, 6.8 mmol) andL-leucylglycine ethyl ester hydrochloride (2.0 g, 7.9 mmol) in 40 mldimethylformamide was added HBT (1.1 g, 7.1 mmol) followed by additionof triethylamine (1.1 ml, 8.0 mmol) and EDC (1.6 g, 8.3 mmol). Thesolution was stirred at room temperature overnight. The triethylaminehydrochloride was removed by filtration and the filtrate concentrated invacuo. The residue was partitioned between ethyl acetate and water andthe organic layer was washed consecutively with dilute hydrochloricacid, water and sodium bicarbonate solution adjusted to pH 8.0. Theethyl acetate was dried over magnesium sulfate. The magnesium sulfatewas removed by filtration and the filtrate concentrated in vacuoyielding 0.86 g of syrupy product. The crude HS-Leu-Leu-Gly-OC₂ H₅ wasused in the next step without further purification.

EXAMPLE 9 Preparation of HS-Leu-Phe-Gly-OC₂ H₅

This mercaptopeptidyl ester was prepared substantially similarly asHS-Leu-Leu-Gly-OC₂ H₅, above, except that an equivalent amount ofL-phenylalanylglycine ethyl ester hydrochloride was used in the couplingreaction instead of L-leucylglycine ethyl ester hydrochloride.

EXAMPLE 10 Preparation of HS-Leu-Ala-Gly-OC₂ H₅

This mercaptopeptidyl ester was prepared substantially similarly asHS-Leu-Leu-Gly-OC₂ H₅, above, except that an equivalent amount ofL-alanylglycine ethyl ester hydrochloride was used in the couplingreaction instead of L-leucylglycine ethyl ester hydrochloride.

EXAMPLE 11 Preparation of Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC₂ H₅

To a solution of Ac-Pro-Leu-Gly-OH (0.7 g, 2.1 mmol), HS-Leu-Leu-Gly-OC₂H₅ (0.6 g, 1.7 mmol) and HBT (0.26 g, 1.7 mmol) in 15 mldimethylformamide was added EDC (0.4 g, 2.1 mmol). The solution wasstirred at room temperature overnight. The solvent was removed in vacuoand the residue partitioned between salt water and ethyl acetate. Theethyl acetate layer was washed consecutively with dilute sodiumbicarbonate in salt (NaCl) water, dilute hydrochloric acid in saltwater, salt water, 1.0 mM cupric sulfate in salt water (to remove anymercapto products) and finally salt water. The ethyl acetate was driedover magnesium sulfate. The magnesium sulfate was removed by filtrationand the filtrate concentrated in vacuo, yielding 0.8 g (73%) glassyresidue of crude thiopeptolide product. A 100 mg sample of this crudeproduct was purified by low pressure C₁₈ column chromatography. Thepurified thiopeptolide, had an acceptable amino acid analysis, aconsistent mass spectrum and a single peak on HPLC analysis. Anal.Calcd. for

C₃₁ H₅₃ N₅ O₈ S.3H₂ O: C, 52.4; H, 8.3; N, 9.9. Found: C, 52.5; H, 7.9;;N, 9.9.

EXAMPLE 12 Preparation of Ac-Pro-Ala-Gly-S-Leu-Phe-Gly-OC₂ H₅

This thiopeptiolide was prepared substantially similary asAc-Pro-Leu-Gly-S-Leu-Phe-Gly-OC₂ H₅, above, except that an equivalentamount of Ac-Pro-Ala-Gly-OH was used in the coupling reaction instead ofAc-Pro-Leu-Gly-OH.

EXAMPLE 13 Preparation of Ac-Pro-Ala-Gly-S-Leu-Ala-Gly-OC₂ H₅

This thiopeptolide was prepared substantially similarly asAc-Pro-Ala-Gly-S-Leu-Phe-Gly-OC₂ H₅, above, except that an equivalentamount of HS-Leu-Ala-Gly-OC₂ H₅ was used in the coupling reacitoninstead of HS-Leu-Phe-Gly-OC₂ H₅.

EXAMPLE 14 Spectrophotometric Assay for Collagenase

Collagenase was prepared from culture media of normal human skinfibroblasts substantially according to the procedure of Stricklin etal., Biochem. 16 1607 (1977). The enzyme (23 μg/ml in 0.5M tris buffer[tris(hydroxymethyl)amino methane], 0.01M CaCl₂, pH 7.5) was activatedby incubating 10 μl samples with one μl of trypsin (10 mg/ml in 1 mMHCl) for twenty minutes at room temperature followed by addition of 20μl of soybean trypsin inhibitor (5 mg/ml in 0.05M tris buffer, 0.01MCaCl₂, pH 7.5).

The activated enzyme was diluted 100 to 2000 fold for use with each ofthe following substrate thiopeptolides:

    Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC.sub.2 H.sub.5,

    Ac-Pro-Leu-Gly-S-Leu-Phe-Gly-OC.sub.2 H.sub.5,

    Ac-Pro-Ala-Gly-S-Leu-Phe-Gly-OC.sub.2 H.sub.5, and

    Ac-Pro-Ala-Gly-S-Leu-Ala-Gly-OC.sub.2 H.sub.5.

The spectrophotometric assay was carried out at substrate concentrationsof from 0.01 mM to 5 mM in 0.05M HEPES buffer(N-2-hydroxyethylpiperizine-N'-2-ethanesulfonic acid) with 0.01M CaCl₂at a pH of 6.5-7.0 and containing 0.5 mM to 1.0 mM 4,4'-dithiodipyridine(or Ellman's Reagent). The total reaction volume was 250 μl including 10to 100 μl of the diluted activated enzyme. The reaction solutions wereplaced in 10 mm pathlength, self-masking microcuvettes and thehydrolysis followed using a Gilford model 250 spectrophotometer at 324 λ(410 λ for Ellman's Reagent) and equipped with a Gilford model 6051recorder. The initial rates were limited to the first 5% of reaction andwere corrected for non-enzymatic hydrolysis, which never exceeded 25% ofthe total reaction at pH 6.5-7.0. The 4,4'dithiodipyridine (or Ellman'sReagent) at 0.5 mM was in large excess under the initial rate assayconditions and was not rate limiting.

The following Table I sets forth the relative rates of hydrolysis in theabove spectrophotometric assay using the above thiopeptolides assubstrates in comparison to the rates of hydrolysis assayed by HighPerformance Liquid Chromatography (HPLC) of two commercially availablesubstrates for collagenase and two other substrates described incopending Application Ser. No. 450,318, cited hereinbefore. HPLC assaywas used for the latter four compounds since they did not contain anychromophores.

                                      TABLE I    __________________________________________________________________________    Comparison of Substrate    Substrate                     Relative Rate    __________________________________________________________________________    Ac--Pro--Leu--Gly--S--Leu--Leu--Gly--OC.sub.2 H.sub.5                                  8800    Ac--Pro--Leu--Gly--S--Leu--Phe--Gly--OC.sub.2 H.sub.5                                  5900    Ac--Pro--Ala--Gly--S--Leu--Phe--Gly--OC.sub.2 H.sub.5                                  2800    Ac--Pro--Ala--Gly--S--Leu--Ala--Gly--OC.sub.2 H.sub.5                                   950    Ac--Pro--Leu--Gly--Leu--Leu--Gly--OC.sub.2 H.sub.5 *                                   250    Ac--Pro--Leu--Gly--Leu--Ala--Gly--OC.sub.2 H.sub.5 *                                   110    DNP--Pro--Leu--Gly--Ile--Ala--Gly--Arg--NH.sub.2 **                                   25    DNP--Pro--Gln--Gly--Ile--Ala--Gly--Gln--D-Arg--OH**                                    5    __________________________________________________________________________     *U.S. Ser. No. 450,318      **Peninsula Laboratories, Inc.

EXAMPLE 15 Enzyme Selectivity for Thiopeptolide

The ability of five proteolytic enzymes other than collagenase to cleavethe synthetic substrate Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC₂ H₅ was testedby hydrolysis and following spectrophotometrically as in Example 14. Thefollowing Table II sets forth the relative selectivity of these enzymesin comparison to vertebrate collagenase.

                  TABLE II    ______________________________________    Enzyme Selectivity    Enzyme            Relative Rate    ______________________________________    Vertebrate Collagenase*                      260    Elastase          12    Kallikrein        4.3    α-Chymotrypsin                      2.0    Trypsin           0.07    Carboxypeptidase Y                      0.03    ______________________________________     *Same as in Example 14

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention and it is intended that all such furtherexamples be included within the scope of the appended claims. Forexample, the terminal ethyl groups in the foregoing thiopeptolides canreadily be converted to other ester groups such as methyl, propyl,benzyl, p-nitrobenzyl or t-butyl ester groups; and other N-protectinggroups such as t-BOC and carbobenzoxy can readily be used in place ofthe acetyl groups or the N-protecting group can readily be removedwithout departing from the basic and novel properties of the invention.

What is claimed is:
 1. A thiopeptolide having activity as a substratefor vertebrate collagenase selected from the group consisting of:

    R-Pro-X-Gly-S-Y-Z-Gly-R.sub.1

wherein R=H or N-protecting group, X=Leu, Ile, Phe, Val, Gln, Ala,Y=Leu, Ile, Phe, Val, Ala, Z=Leu, Ile, Phe, Val, Gln, Ala, R₁ =terminalamide, carboxyl or ester group, and the pharmaceutically acceptablesalts thereof.
 2. The thiopeptolide of claim 1 in whichX=Leu or Ala,Y=Leu, and Z=Leu, Ala or Phe.
 3. The thiopeptolide of claim 2 having thestructure

    Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC.sub.2 H.sub.5.


4. The thiopeptolide of claim 2 having the structure

    Ac-Pro-Leu-Gly-S-Leu-Phe-Gly-OC.sub.2 H.sub.5.


5. The thiopeptolide of claim 2 having the structure

    Ac-Pro-Ala-Gly-S-Leu-Phe-Gly-OC.sub.2 H.sub.5.


6. The thiopeptolide of claim 2 having the structure

    Ac-Pro-Ala-Gly-S-Leu-Ala-Gly-OC.sub.2 H.sub.5.